Randomized clinical trial testing the efficacy and safety of 0.5% colchicine cream versus photodynamic therapy with methyl aminolevulinate in the treatment of skin field cancerization:

The primary clinical manifestation of skin field cancerization is the presence of actinic keratoses (AKs). Current treatments for AKs related to skin field cancerization include photodynamic therapy (PDT) and colchicine.

S T U D Y P R O T O C O L Open Access

Randomized clinical trial testing the

efficacy and safety of 0.5% colchicine

cream versus photodynamic therapy with

methyl aminolevulinate in the treatment of

skin field cancerization: study protocol

Anna Carolina Miola*, Eliane Roio Ferreira, Luciana Patricia Fernandes Abbade, Juliano Vilaverde Schmitt

and Helio Amante Miot

Abstract

Background: The primary clinical manifestation of skin field cancerization is the presence of actinic keratoses (AKs) Current treatments for AKs related to skin field cancerization include photodynamic therapy (PDT) and colchicine The objective of this study is to evaluate the efficacy and safety of 0.5% colchicine cream versus PDT with methyl aminolevulinate (MAL-PDT) in the treatment of skin field cancerization

Methods: In a randomized controlled and open clinical trial with a blind histopathological and immunohistochemical analysis, 36 patients with up to 10 AKs on their forearms will be included from the outpatient clinic The forearms will

be randomized into two groups, clinically evaluated and biopsied for histopathology and immunohistochemistry (p53 and Ki67) One forearm will be treated with 0.5% colchicine cream for 10 days, and the other forearm will receive one session of MAL-PDT; the forearms will subsequently be reassessed clinically and histologically after 60 days (T60) of treatment The primary endpoint will be the point of complete clearance of AKs in T60 The sample size will enable a detection in the reduction of over 10% in AK counts between the groups with power of 0.9 and an alpha of 0.05, accounting for an estimated dropout rate of 10%, resulting in 36 patients (72 forearms) All participants included

in the randomized study will be part of the analysis, and the final outcomes of any dropouts will be the value of their last visit (LOCF) The statistical analysis will be performed using SPSS 22.0, and ap value < 5% will be considered

to be significant

Discussion: It is expected that colchicine will be superior to MAL-PDT in reducing AKs and in the skin field cancerization, and there will be good tolerability in both groups Colchicine intervention is novel in that it provides a new alternative to MAL-PDT Moreover, this drug is inexpensive that may be a potential treatment of skin field cancerization that can be prescribed in public health systems with good results

Trial registration: The trial is registered in Brazilian Registry for Clinical Trials (Registration number:RBR-8y3sj9, date assigned May 4, 2016, retrospectively registered)

Keywords: Actinic keratoses, Colchicine, Methyl aminolevulinate, Skin cancer, Skin field cancerization,

Photodynamic therapy

* Correspondence: anna_fmrp@yahoo.com.br

Department of Dermatology and Radiotherapy, Botucatu Medical School,

Universidade Estadual Paulista, UNESP, São Paulo, Brazil

© The Author(s) 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver

Actinic keratosis

Actinic keratosis (AK), the most common premalignant

lesion, affects sun-exposed areas as a result of chronic

exposure to UVR [1] Individuals with skin types I and II

of the Fitzpatrick classification with excessive sun exposure,

advanced age or immunosuppression comprise the highest

risk group [2]

AK lesions may be macular or papular injuries with

adherent and rough scales, a color varying from yellow

to brown, and a size of approximately 0.5 to 1 cm, and

they can converge on plaques AKs occur primarily in

the areas most exposed to sunlight, such as the face,

scalp, forearms and backs of hands [3]

Infiltration, rapid growth, bleeding and ulceration may

indicate the progression of AK to squamous cell

carcin-oma (SCC) [4], the incidence of which varies between 0

025% and 20% of patients with AKs per year [5] AKs

are the most obvious clinical manifestation of skin field

cancerization activity and are used to quantify this activity

due to the absence of other quantification methods

Field cancerization

First described in 1953 by Slaughter, field

canceriza-tion refers to an apparently normal tegument area

with subclinical and multifocal changes, composed of

genetically altered cells [6] The concept of skin field

cancerization suggests that the apparently normal

skin adjacent to actinic keratoses (AKs) is the basis

for the clonal expansion of genetically altered cells

This phenomenon can explain the appearance of

new AKs or other nonmelanoma skin tumors in the

same area, as well as the local recurrence of tumors

considered to be completely excised by

histopatho-logical analysis [7]

Recently, skin field cancerization has been extensively

studied, due to its clinical importance The alteration of

adjacent cells makes it necessary to treat the area as a

whole [8] to prevent recurrence, the appearance of new

lesions and the progress of existing lesions

Treatment modalities that aim to stabilize the activity

of skin field cancerization are warranted because they

may decrease morbidity and mortality in the exposed

population and prevent future neoplastic and

preneo-plastic injuries [9]

Available treatments

The purpose of treating AKs and skin field cancerization

is to mitigate the risk of the AKs progressing to

malig-nant lesions and cancer development [10] Several

treat-ment modalities exist, which are chosen according to

the following: size of the area, number of lesions,

im-munosuppression or other co-morbidities, cost of

treat-ment, tolerability and patient adherence [11]

Cryotherapy with liquid nitrogen, which is the most common AK treatment, is well tolerated, easily access-ible and shows good clinical and histopathological re-sults (clinical response of 83% with a freezing time longer than 20 s) [12] Discomfort during the application and post-procedure hypochromia can occur and may persist [13] Cryotherapy treats only visible lesions, making

it a good treatment for AKs, but this therapy does not treat skin field cancerization [14]

Another treatment option is 5-fluorouracil (5FU), a topical chemotherapy and antimetabolic drug that dis-rupts DNA synthesis by thymidylate synthase binding [15] At concentrations ranging from 0.5 to 5%, 5FU has been shown to produce good therapeutic responses in treating AK, but aggressive side effects can occur, such

as erythema, pain, swelling and ulceration [16] 5FU im-proves the overall appearance of skin, decreases p53 ex-pression and stabilizes skin field cancerization even after six months of treatment [17]

Imiquimod is a topical immunomodulator toll-like receptor 7 agonist [18], which stimulates local immunity with consequent apoptosis by injury Imiquimod is com-mercially available in 3.75% and 5% formulations Unlike 5-FU, which may cause erythema, edema and local vari-able discomfort, treatment with 5% imiquimod, applied three times per week for eight weeks, has been shown to cause an 82% reduction of AKs with only slight moder-ate local irritation reported [19]

Ingenol mebutate is a Euphorphia peplus-derived ester with two distinct forms of action: either direct cell death induction through changes in the mitochondrial and plasma membranes of dysplastic cells or local immune response induced by proinflammatory cytokines and massive neutrophil infiltration [20] The application of 0 05% ingenol mebutate twice a day for two consecutive days showed a complete response rate of 71% Moreover, with minimal systemic absorption, ingenol mebutate has tolerable side effects, such as erythema, crusting and pain [21]

Diclofenac is a non-steroidal anti-inflammatory drug that inhibits the enzyme cyclooxygenase type 2, the ac-tivity of which is increased in AKs and non-melanoma skin tumors [22] When compared to a placebo, topical diclofenac at 3% in 2.5% hyaluronic acid gel used for

90 days demonstrated an efficacy of 50% compared with 3% in the placebo group Adverse events, such as erythema, pruritus, contact dermatitis and skin xerosis, are rare; how-ever, Diclofenac requires a long treatment period for an adequate response [23]

In addition to the abovementioned treatments, other treatments for skin field cancerization are known, such

as resiquimod, tretinoin [24], colchicine and photo-dynamic therapy (PDT); the latter two are included in this study protocol

Photodynamic therapy

Photodynamic therapy (PDT) is a treatment modality

that induces cytotoxicity of the proliferating cells of a

tumor through a light emission source; this activity is

lo-calized through a photosensitizing agent which can be

oral or topical [25]

In dermatology, PDT is used for the treatment of

pre-malignant and non-melanoma skin tumors, such as AKs,

superficial basal cell carcinoma and Bowen disease, or

SCC in situ [26] PDT is also used for skin rejuvenation

and to treat inflammatory skin diseases, such as

psoria-sis, warts, morphea and Kaposi’s sarcoma [27]

For the implementation of PDT, patients should first

be exposed to a photosensitizing agent, usually a topical

one The following two photosensitizing agents are

li-censed: 5-aminolevulinic acid (ALA-PDT) and its

lipo-philic derivative, methyl aminolevulinate (MAL) [28]

MAL is preferred for its non-saturable absorption

mech-anism, which results in greater penetration and thus

greater uptake by neoplastic cells [29]

MAL is a precursor of protoporphyrin IX, which

gen-erates reactive oxygen in dysplastic keratinocytes when

exposed to a light source with the appropriate

wave-length [30]

MAL is topically administered in a 16% cream

formu-lation and maintained under occlusion for 3 h before the

light source exposure, which can originate from one of

three sources: a broad-spectrum lamp, a light-emitting

diode lamp (LED) or a laser [31]

During PDT, MAL is activated and generates reactive

oxygen During this process, MAL is converted from its

resting state to an active form called singlet, which has a

short half-life Due to the presence of singlet, cells start

to undergo changes in their membranes, which affect

the permeability and transport of substances and cause

toxicity of cytoplasmic organelles, inducing apoptosis in

cancer cells [32]

PDT has shown response rates close to 90% in thin

and moderate AKs on the face and scalp, and that

re-sponse has been shown to be sustained for one year in

39 to 63% of patients with only one session and 78% of

pa-tients with two sessions In addition, a good tolerability,

minimal adverse effects with only a short duration, and

very satisfactory aesthetic results have been shown [33]

Colchicine

Colchicine is an antimitotic agent that binds to

micro-tubular proteins and interferes with the mitotic spindle

activity, interrupting mitosis at metaphase [34,35]

Col-chicine also inhibits the chemotaxis of

polymorpho-nuclear leukocytes Side effects can include abdominal

pain, nausea, vomiting, agranulocytosis and aplastic

anemia Topical application can cause erythema, edema,

crusts and vesicles in the treated area Toxicity is serious

and can occur with any route of administration Colchi-cine has a slow elimination, preferably through the intes-tine and kidney [36]

In dermatology, colchicine has several therapeutic ap-plications, both systemic and topical, such as treating the following: psoriasis vulgaris, generalized pustular psoriasis, pustulosis palmar plantar, small vessel vascu-litis, type II reaction of leprosy, Behcet’s disease and others [37]

Topical colchicine was first used for treating AKs in

1968 by Marshall [38], and few studies on colchicine have been conducted since

Colchicine has been studied at concentrations of 0.5 and 1% in a gel and has been demonstrated to reduce AKs by 77% with satisfactory after-treatment aesthetic results [39]

However, there have been few studies that have sys-tematized the dosage and duration of treatment with colchicine, and no studies comparing the efficacy of col-chicine and photodynamic therapy with methyl aminole-vulinate (MAL), a treatment modality that has been tested in more clinical trials for skin field cancerization Therefore, the present study was undertaken

Objectives

The objective of this study is to evaluate the efficacy and safety of 0.5% colchicine cream versus PDT with methyl aminolevulinate (MAL-PDT) in treating skin field cancerization in patients with multiple actinic keratoses

Research question and hypotheses

Is 0.5% colchicine cream more effective than MAL-PDT treatment for the complete clearance of AKs (primary endpoint), partial clearance of AKs, histological modifi-cation of the atypical profile, p53 and Ki67 expression and tolerability (secondary outcomes) after 60 days of treatment?

Main hypotheses are as follows:

1 Colchicine and MAL-PDT (as proposed schemes) will both reduce the number of AKs and modify skin field cancerization histology

2 Colchicine and MAL-PDT (proposed schemes) will both show good tolerability and safety in skin field cancerization treatment

3 Colchicine will be superior to MAL- PDT (proposed schemes) in AK reduction and in modifying skin field cancerization histology

Methods/Design

This randomized clinical trial will be controlled and open with blind histopathological and immunohisto-chemical analyses Thirty-six patients with 3 to 10

AKs on each forearm will be treated with colchicine

cream on one forearm and MAL-PDT on the other

forearm Patients will be evaluated for the number of

AKs and the expression of Ki67 and p53 before and

after the treatments

This study was approved by the Medical Ethics

Committee of the institution and recorded in REBEC

under RBR-8y3sj9

Selection of participants

Inclusion criteria

 Age over 18 years for both sexes

 Participants who present three to ten lesions

clinically compatible with AKs on each forearm,

bilaterally, who have not undergone treatment for

AKs or field cancerization in the last six months

Exclusion criteria

 Number of lesions is less than three or greater than

ten on each forearm

 Selected treatment area has atypical clinical

appearance or other extensive skin disease affecting

the forearms

 Current clinical diagnosis or evidence of any medical

condition that expose the patient to increased risk

or interferes with the safety or efficacy of the

proposed treatment

 Presence of hypersensitivity or allergy to any of the

substances under study

 Patients using any topical or systemic

immunosuppressive substance, oral retinoid or other

local treatments (corticosteroids, anti-inflammatories,

retinoids)

 Immunocompromised individuals

 Coagulation disorders

 Pregnancy suspected or confirmed

 Women of childbearing potential not using

contraception

 Women who are breast-feeding

Discontinuation criteria

 Withdrawal of consent

 Presence of infection (erysipelas, cellulitis or abscess)

in the monitored treatment areas In such cases,

research subject should be properly treated as a

routine service

 Patients using methods of treatment different than

those proposed in this study

 Serious adverse events, according to investigator

 Pregnancy during follow-up

Participant recruitment

Thirty-six patients from the dermatology department of the Botucatu Medical School will be recruited Patients will be informed regarding the possibility of participating

in this processing study during their dermatological consultation All of the patients will sign a consent form before enrollment, and they will be scheduled for out-patient treatment for the study itself without loss of follow-up after study participation

Randomization and concealment of allocation of treatments

Patient forearms will be randomized by computer simu-lation (block randomization) for treatment with 0.5% colchicine cream or MAL-PDT Inclusion will be held consecutively to the scheduling of patients, regardless of the randomization list

Interventions

The study units of analysis are forearms, the treatment

of which will be randomized to groups designated as PDT-MAL and COL

In the COL group, patients will be recommended to use 0.5% cream twice daily for 10 days on the forearm Adherence to the treatment will be monitored by phone calls In the MAL-PDT group, forearms will receive one MAL-PDT session [40], administered by the same re-searcher The protocol for the MAL-PDT session is as follows: AK lesions will first be curetted Next, a layer of 16% methyl aminolevulinate will be applied, and the forearm will be occluded for 3 h while covered by PVC film and aluminum foil Next, the forearm will be illumi-nated with a light emitting diode (LED) with a wave-length of 630 nm at a distance of five to eight centimeters for eight minutes All patients will wear safety glasses to protect their eyes

The study will last 60 days for each patient During this time, patients will be instructed to apply SPF 30 broad spectrum sunscreen (UVA and UVB) to their forearms

The area of AK count, evaluation and punch 3.0 mm biopsy will be standardized The assessed area will have

a distal limit as a line at the metacarpophalangeal joints and a proximal limit as a line extending from antecubital fossa to the lateral epicondyle of the anterior side of the forearms Biopsies will be taken in central region of the middle third of each assessed area

Participants will be assessed at the following times: T0 - inclusion, randomization, clinical evaluation (AK count), skin biopsy (bilateral) and beginning of treat-ment; T15 - assessment of adverse effects and toler-ability; T60 - clinical evaluation (AK count), reassessment

of adverse effects and tolerability, and bilateral skin biopsy [see Fig.1]

Primary endpoint:

 Completeclearance rate of AKs when treated with

colchicine and MAL-PDT in T60 secondary

outcomes

 Partialclearance rate of AKs when treated with

colchicine and MAL-PDT, comparing T0 to T60

 Modification of histological atypia profile between

treatments, comparing T0 to T60

 Modification of the expression of p53 and Ki67

between treatments, comparing T0 to T60

 Adverse effects related to the proposed treatment:

impact of local events and severity

 Emergence of non-melanoma skin tumors

 Tolerability assessed by the patient reporting

adverse effects, preferably between treatments

Data analysis

The main study endpoints will be the longitudinal

evalu-ation of AK count, histological changes and proliferevalu-ation

of scores (p53 and Ki67)

Diagnosis of AKs will be performed based on a clinical

examination by a certified dermatologist Counts of AKs

will be performed twice by same researcher after the

le-sions are marked with a felt-tip pen, with no distinction

being made between visible and palpable forms [41]

Epithelial atypia will be evaluated (T0 and T60)

based on H&E staining according to KIN classification

(Keratinocyte Intraepithelial Neoplasia) in three

cen-tral fields in interfollicular areas, and the investigator

will not know which group the samples are from

For immunohistochemistry, histological sections of

4 μm thickness will be mounted on silanized slides and

subjected to immunohistochemical staining for detection

of Ki67 (Cell Sig Tech., Inc., Danvers, MA, USA, mouse mAb IgG1, #9449) or p53 (Cell Sig Tech., Inc., Danvers,

MA, USA, mouse mAb Ig2b, #48818) After sections deparaffinization and rehydration will be performed the endogenous peroxidase blockade with 3% H2O2, followed by heat-induced antigen retrieval and protein blockade by treatment with milk The sections will be incubated overnight at 4 °C with the primary antibody diluted 1:300 or 1:150 for the Ki67 and p53 respectly Signal amplification will be performed by biotin-free poly-mer detection system using the MACH 4® universal HRP-Polymer kit (Biocare Medical, Pike Lane Concord, CA, USA) according to the manufacturer’s instructions Lastly, 3–3′ diaminobenzedine will be used as a chromogenic substrate and the sections will be counterstained with Harris hematoxylin Epithelial expression of p53 and Ki67 will be evaluated (T0 and T60) based upon the HSCORE, calculated in three central fields (40×), of the sampled epi-thelium, and the investigator will not know from which group the samples are obtained

Patients will be questioned at T15 and T60 regarding whether they would accept this treatment again and which treatment they preferred Any AKs remaining after the study will be treated with cryotherapy with liquid nitrogen

Sample size

The sample size was chosen to detect a reduction in AKs of over 10% between groups and a standard de-viation of equivalent differences A power of 0.9 and

an alpha of 0.05 were adopted, and the dropout rate was estimated to be 10%, resulting in 36 patients (72 forearms)

Fig 1 Flow chart of the study (CONSORT)

Statistical analysis

All participants included in study will be randomized

in the ITT (intention to treat) group Data analysis

will be performed for the ITT population following

the generalized linear mixed-effects model The final

outcomes for dropouts will be the value of last visit

(LOCF)

Regarding the group descriptions, categorical variables

will be presented as the absolute number and

percent-age Continuous variables will be assessed for normality

using the Kolmogorov-Smirnov test (Lilliefors) and

rep-resented by means and standard deviations or medians

and quartiles (p25-p75)

The AK count and the analysis of the histopathological

data will be compared with respect to time and group

(over time) using the linear mixed-effects model with

ro-bust covariance matrix and probability adjustment for

each distribution

Data will be tabulated in Microsoft Excel 2013, and

statistical analyses will be performed using SPSS 22.0; a

p value < 5% will be considered to be significant

Discussion

Dissemination of the results of this study should

reach the scientific community through conferences

and scientific publications, as well as the institutional

research and ethics committee where the work was

developed

Results of this study may help to reduce the costs

of skin field cancerization treatment and expand new

treatment strategies The primary limitation of this

trial is the impossibility of clinical blinding; however,

the histopathological and immunohistochemical

ana-lyses will be blind, and the AK count prior to

treat-ment will also be blind, thereby reducing biases

Based upon the results of this study,

dermatolo-gists may approach new treatments, and skin field

cancerization treatment may lead to a lower

inci-dence of keratinocytic cancer, thereby directly

bene-fiting society

Abbreviations

5-FU: 5-fluorouracil; AK: Actinic keratosis; ALA: 5-aminolevulinic acid;

LED: Light emitter diode; MAL: Methyl-aminolevulinate; MAL: With

methyl-aminolevulinate; PDT: Photodynamic therapy; PDT: Photodynamic therapy;

R: Radiation; UV: Ultraviolet; UV: Ultraviolet

Acknowledgements

Not available.

Funding

This research received no specific grant from any funding agency in the

public, commercial or not-for-profit sectors.

Availability of data and materials

Not available.

Authors ’ contributions ACM was involved in the bibliographic review, study design, testing of the data extraction form, and writing the initial draft HAM was responsible for the study conception, designing the review and critically reviewing the final draft EFR was involved in the bibliographic review and in testing the data extraction form LPFA and JS contributed to improvements in the manuscript and critically revised the final draft All authors contributed to the protocol and approved the final manuscript.

Ethics approval and consent to participate This study was approved by the Medical Ethics Committee of the institution

in 7 dec, 2015 (n 1.356.401) and consent to participate is attached below Consent for publication

Not available.

Competing interests The authors declare that they have no competing interests.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Received: 26 September 2016 Accepted: 21 March 2018

References

1 Berking C, Takemoto R, Binder RL, Hartman S, Ruiter TJ, Gallagher PM, Lessin SR, Herlyn M Photocarcinogenesis in human adult skin grafts Carcinogenesis 2002;23:181 –7.

2 Salasche SJ Epidemiology of actinic keratoses and squamous cell carcinoma J Am Acad Dermatol 2000;42:4 –7.

3 Rossi R, Mori M, Lotti T Actinic keratosis Int J Dermatol 2007;46:895 –904.

4 Moy RL Clinical presentation of actinic keratoses an squamous cell carcinoma J Am Acad Dermatol 2000;42:8 –10.

5 Marks R, Rennie G, Selwood TS Malignant transformation of solar keratoses

to squamous cell carcinoma in the skin: a prospective study Lancet 1988;1:

795 –7.

6 Slaughter DP, Southwick HW, Smejkal W “Field cancerization” in oral stratified squamous epithelium Cancer (Phila.) 1953;6:963 –8.

7 Braakhuis BJ, Tabor MP, Kummer JA, Leemans CR, Brakenhoff RH A genetic explanation of Slaughter ’s concept of field cancerization evidence and clinical implications Cancer Res 2003;63:1727 –30.

8 Dakubo GD, Jakupciak JP, Birch-Machin MA, Parr RL Clinical implications and utility of field cancerization Cancer Cell Int 2007;12:1 –12.

9 Torezan LAR, Festa-Neto C Cutaneous field cancerization: clinical, histopathological and therapeutic aspects An Bras Dermatol 2013;88(5):

779 –91.

10 Berman B, Cohen DE, Amini S What is the role of field-directed therapy in the treatment of actinic keratosis? Part 1: overview and investigational topical agents Cutis 2012;89(5):241 –50.

11 De Berker D, McGregor JM, Hughes BR Guidelines for the management of actinic keratoses Br J Dermatol 2007;156(2):222 –30.

12 Oliveira MC, Pinto CAL, Pinto JCCL, Trevisan F, Xavier CA Histopatological analysis of the therapeutic response to cryotherapy with liquid nitrogen in patients with multiple actinic keratosis An Bras Dermatol 2015;90(3):384 –9.

13 Poziomczyk CS, Koche B, Dornelles MA, Dornelles SIT Pain evaluation in the cryosurgery onf actinic keratosis An Bras Dermatol 2011;86(4):645 –50.

14 Szeimies RM, Karrer S, Radakovic-Fijan S, Tanew A, Calzavara-Pinton PG, Zane C, et al Photodynamic therapy using topical methyl 5-aminolevulinate compared with cryotherapy for actinic keratosis: a prospective, randomized study J Am AcadDermatol 2002;47:258 –62.

15 Bagatin E 5-fluorouracil for actinic keratoses Exp RevDermatol 2010;5:131 –9.

16 Labandeira J, Pereiro M, Valdés F, Toribio J Intermittent topical 5-fluorouracil

is effective without significant irritation in the treatment of actinic keratoses but prolongs treatment duration Dermatol Surg 2004;30:517 –20.

17 Okada C, Guimarães Z, Miot HA Five percent 5-fluorouracil in a cream or for superficial peels in the treatment of advanced photoaging of the forearms:

a randomized comparative Study Dermatol Surg 2014;40(6):610-7.

18 Dockrell DH, Kinghorn GR Imiquimod and resiquimod as novel immunomodulators J Antimicrob Chemother 2001;48(6):751 –5.

19 Salasche SJ, Levine N, Morrison L Cycle therapy of actinic keratoses of the

face and scalp with 5% topical imiquimod cream: an open-label trial J Am

Acad Dermatol 2002;47:571 –7.

20 Rosen RH, Med MM, Gupta AK, Tyring SK Dual mechanism of action of

ingenol mebutate gel for topical treatment of actinic keratoses: rapid lesion

necrosis followed by lesion-specific immune response J Am Dermatology

Elsevier Inc 2012;66(3):486 –93.

21 Lebwohl M, et al Ingenol mebutate gel for actinic keratosis N Engl J Med.

2012;366(11):1010 –9.

22 Berman B, Villa AM, Ramirez CC Mechanisms of action of new treatment

modalities for actinic keratosis J Drugs Dermatol 2006;5:167 –73.

23 Wolf JR, Taylor JR, Tschen E Topical 3% diclofenac in 2,5% hyaluronan gel in

the treatment of actinic keratoses Int J Dermatol 2001;40:709 –13.

24 Ianhez M, Fleury LFF Jr, Miot HA, Bagatin E Retinoids for prevention and

treatment ofactinic keratosis An Bras Dermatol 2013;88(4):585 –93.

25 Torezan LA, Niwa AB, Neto CF Photodynamic therapy in dermatology: basic

principles An Bras Dermatol 2009;84(5):445 –59.

26 Ozog DM, Rkein AM, Fabi SG, Gold MH, Goldman MP, Lowe NJ, et al.

Photodynamic Therapy: A Clinical Consensus Guide Dermatol Surg 2016;

42(7):804 –27.

27 Torezan L, Niwa ABM, Festa NC Terapia fotodinamica em dermatologia:

principios basicos e aplicacoes An Bras Dermatol 2009;84(5):445 –59.

28 Szeimies RM, Karrer S, Sauerwald A, Landthaler M Photodynamic therapy

with topical application of 5-aminolevulinic acid in the treatment of actinic

keratoses: an initial clinical study Dermatology 1996;192:246 –51.

29 Morton CA, Szeimies R-M, Bratheen LR Update on topical photodynamic

therapy for skin cancer JEADV 2014;6:26 –34.

30 Angell-Petersen E, Sorensen R, Warloe T, Soler AM, Moan J, Peng Q, et al.

Porphyrin formation in actinic keratosis and basal cell carcinoma after

topical application of methyl 5-aminolevulinate J Invest Dermatol 2006;126:

265 –71.

31 Calzavara-Pinton PG, Venturini M, Sala R Photodynamic therapy: update

2006 Part 1: photochemistry and photobiology J Eur Acad Dermatol

Venereol 2007;21:293 –302.

32 Nelson JS, McCullough JL, Berns MW Principles and applications of

photodynamic therapy in dermatology In: Ardnt KA, Dover JS, Olbright SM,

editors Lasers in cutaneous and aesthetic surgery Philadelphia:

Lippincot-Raven; 1997 p 349 –82.

33 Nestor MS, Gold MH, Kauvar ANB, Taub AF, Geronemus RG, Ritvo EC, et al.

The use of phodynamic therapy in dermatology: results of a consensus

conference J Drugs Dermatol 2006;5:140 –54.

34 Malkinson FD Colchicine: new uses of an old, old drug Arch Dermatol.

1982;118:453 –7.

35 Molad Y Update on colchicine and its mechanism of action Curr Rheumatol

Rep 2002;4:252 –6.

36 Levy M, Spino M, Read SE Colchicine: a state of the art review.

Pharmacotherapy 1991;11:196 –211.

37 Sullivan TP, King LE, As B Colchicine in dermatology J Am Acad Dermatol.

1998;39:993 –9.

38 Marshall J Treatment of solar keratoses with topically applied cytostatic

agents Br J Dermatol 1968;80:540 –2.

39 Akar A, Bülent Ta ştan H, Erbil H, Arca E, Kurumlu Z, Gür AR Efficacy and

safety assessment of 0.5% and 1% colchicine cream in the treatment of

actinic keratoses J Dermatolog Treat 2001;12(4):199 –203.

40 Braathen LR, Morton CA, Basset-Seguin N, Bissonnette R, Gerritsen MJP,

Gilaberte Y, et al Photodynamic therapy for skin field cancerization: an

international consensus International Society for Photodynamic Therapy in

dermatology JEADV 2012;26:1063 –6.

41 Ianhez M, Fleury Junior LF, Bagatin E, Miot HA The reliability of counting

actinic keratosis Arch Dermatol Res 2013;305(9):841 –4.

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